Search

Mutations in the cardiac ryanodine receptor Ca(2+) release channel, RyR2, underlie catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited life-threatening arrhythmia. CPVT is triggered by spontaneous RyR2-mediated sarcoplasmic reticulum (SR) Ca(2+) release in response to SR Ca(2+) overload during beta-adrenergic stimulation. However, whether elevated SR Ca(2+) content--in the absence of protein kinase A activation--affects RyR2 function and arrhythmogenesis in CPVT remains elusive.Isolated murine ventricular myocytes harbouring a human RyR2 mutation (RyR2(R4496C+/-)) associated with CPVT were investigated in the absence and presence of 1 micromol/L JTV-519 (RyR2 stabilizer) followed by 100 micromol/L ouabain intervention to increase cytosolic [Na(+)] and SR Ca(2+) load. Changes in membrane potential and intracellular [Ca(2+)] were monitored with whole-cell patch-clamping and confocal Ca(2+) imaging, respectively. At baseline, action potentials (APs), Ca(2+) transients, fractional SR Ca(2+) release, and SR Ca(2+) load were comparable in wild-type (WT) and RyR2(R4496C+/-) myocytes. Ouabain evoked significant increases in diastolic [Ca(2+)], peak systolic [Ca(2+)], fractional SR Ca(2+) release, and SR Ca(2+) content that were quantitatively similar in WT and RyR2(R4496C+/-) myocytes. Ouabain also induced arrhythmogenic events, i.e. spontaneous Ca(2+) waves, delayed afterdepolarizations and spontaneous APs, in both groups. However, the ouabain-induced increase in the frequency of arrhythmogenic events was dramatically larger in RyR2(R4496C+/-) when compared with WT myocytes. JTV-519 greatly reduced the frequency of ouabain-induced arrhythmogenic events.The elevation of SR Ca(2+) load--in the absence of beta-adrenergic stimulation--is sufficient to increase the propensity for triggered arrhythmias in RyR2(R4496C+/-) cardiomyocytes. Stabilization of RyR2 by JTV-519 effectively reduces these triggered arrhythmias.

In the cardiac dyad, sarcolemmal L-type Ca(2+) channels (LCCs) and sarcoplasmic reticulum (SR) Ca(2+) release channels (RyR) are structurally in close proximity. This organization provides for an efficient functional coupling, tuning SR Ca(2+) release for optimal contraction of the myocyte. Given that LCC are regulated by the prevailing [Ca(2+)], this structural organization is the setting for feedback mechanisms and crosstalk. A defective coupling of Ca(2+) influx via LCC to activation of RyR has been implicated in reduced SR Ca(2+) release in heart failure. Both functional changes in LCC properties and structural re-organization of LCC in T-tubules could be involved. LCC are regulated by cytosolic Ca(2+), and crosstalk with SR Ca(2+) handling occurs on a long-term basis, i.e. during steady-state changes in heart rate, on an intermediate-term basis, i.e. on a beat-to-beat basis during sudden rate changes, and on a very short- or immediate-term basis, i.e. during a single heartbeat. We review the properties and consequences of these different feedback mechanisms and the changes in heart failure and cardiac hypertrophy that have thus far been studied.

Objective Reactive oxygen species (ROS) play an ambivalent role in cardiomyocytes: low concentrations are involved in cellular signaling, while higher concentrations contribute to cellular injury. We studied ROS formation during increases in contraction frequency in isolated cardiomyocytes. Methods Rat ventricular cardiomyocytes were loaded with dichlorodihydrofluorescein and electrically stimulated (37 °C). ROS formation was assessed by the rate of oxidation-dependent fluorescence increase (OxR). Oxygen consumption (VO2) and NAD(P)H autofluorescence were measured in parallel experiments. Results Increases in contraction frequency were accompanied by an increase in VO2 and a decrease in NAD(P)H fluorescence. OxR increased to 124±4%, 146±8%, 204±25% and 256±29% of OxR at baseline during 1, 2, 3 and 4 Hz stimulation, and subsequently returned to baseline values with 0.2 Hz. The OxR increase was dose-dependently inhibited by the antioxidant NAC (10 and 100 mM), but unaffected by the NO synthase inhibitor l-NAME (200 μM and 10 mM). The OxR increase was attenuated when myosin ATPase activity was inhibited by butanedione monoxime (BDM; 5 mM). Conclusion Increased contraction frequency induces ROS formation in rat cardiomyocytes.

Urocortin II (UcnII), a peptide of the corticotropin-releasing factor (CRF) family, exerts profound actions on the cardiovascular system. Direct effects of UcnII on adult cardiomyocytes have not been evaluated before. Our aim was to characterize functional effects of UcnII on cardiomyocytes and to elucidate the underlying signaling pathway(s) and cellular mechanisms.Rabbit ventricular cardiomyocytes were stimulated at 0.5 Hz (22-25 degrees C). Unloaded cell shortening (FS, edge detection), [Ca(2+)](i) transients (Fluo-4), and L-type Ca(2+) currents (I(Ca), whole-cell patch clamping) were measured. Sarcoplasmic reticulum (SR) Ca(2+) load was assessed by rapid application of caffeine (20 mmol/L).UcnII increased cell shortening and accelerated relaxation in a time- and concentration-dependent manner (EC(50): 10.7 nmol/L). The inotropic effect of UcnII was maximal at 100 nmol/L (35%+/-11% increase in FS, n=8, P0.05). The inotropic and lusitropic actions of UcnII were largely eliminated by inhibition of CRF(2) receptors (10 nmol/L antisauvagine-30, n=5) or protein kinase A (PKA, 500 nmol/L H-89, n=5). UcnII increased [Ca(2+)](i) transient amplitude (by 63%+/-35%, n=7, P0.05) and decreased the time constant for decay (from 800+/-63 to 218+/-27 ms, n=7, P0.001). UcnII also increased SR Ca(2+) load (by 19%+/-7%, n=7, P0.05) and fractional Ca(2+) release (from 57%+/-7% to 98%+/-2%, n=7, P0.01). I(Ca) was augmented by 32.7%+/-10.0% (n=9, P0.05) and the I(Ca)-V relationship was shifted by -15 mV during UcnII treatment.UcnII exerts positive inotropic and lusitropic effects in cardiomyocytes via CRF(2) receptor-mediated stimulation of PKA which augments I(Ca) and SR Ca(2+) load to increase SR Ca(2+) release and [Ca(2+)](i) transients.

Purpose: Spontaneous sarcoplasmic reticulum (SR) Ca2+ release through defective cardiac ryanodine receptor (RyR2) promotes contractile dysfunction and myocardial remodeling in response to pressure overload. We tested the hypothesis that RyR2 stabilizer K201 attenuates the SR Ca2+ leak and ameliorates pressure overload-induced cardiac remodeling in mice carrying RyR2 gain-of-function mutation. Methods: RyR2R4496C+/- knock-in mice (R4496C) and their wild-type (WT) littermates underwent surgery without (Sham) or with transaortic banding (TAB). Changes in Ca2+ spark frequency (as a measure for the SR Ca2+ leak) were monitored by means of confocal Ca2+ imaging (Fluo-4/AM, linescan mode) in isolated ventricular cardiomyocytes (1 week post-surgery) under control conditions and after the pre-incubation (≥ 1-hour) with 300 nmol/L K201 or 1 mmol/L dantrolene (positive control). K201 (12 mg/kg/day) or DMSO (vehicle) was administered in R4496C-TAB mice for 4 weeks via osmotic minipumps. K201 plasma concentration was determined by using high performance liquid chromatography. Structural dimensions and function of the left ventricle (LV) were assessed by transthoracic echocardiography 1 and 3 weeks after surgery. Data are shown as mean±S.E.M. Results: K201 markedly reduced the increase in Ca2+ spark frequency at matched SR Ca2+ load in R4496C-TAB (83.9±8.6 pL-1s-1 vs. 332.7±42.9 pL-1s-1) and WT-TAB cells (82.2±6.5 pL-1s-1 vs. 190.1±27 pL-1s-1) as well as R4496C-Sham cells (72.1±7.5 pL-1s-1 vs. 173.6±20.9 pL-1s-1, all P

Background: Heart failure with preserved ejection fraction (HFPEF) is increasingly common but the established heart failure (HF) drugs are not effective. The underlying cellular mechanisms are incompletely understood. Therefore we investigated cardiomyocyte function and intracellular Ca2+ homeostasis in a model of HFPEF. Methods: Young male Wistar rats were subjected to subtotal nephrectomy (NXT) or sham operation (SOP). Serial blood/urine samples, echocardiography and pressure-volume loops at 8 and 24 weeks were performed. After sacrifice, left ventricular (LV) hypertrophy and NCX function (Caffeine induced Ca2+ transient, TAU) and protein expression (Western blot) were determined. Cardiomyocyte function (Ca2+ transients, sarcoplasmic reticulum (SR) diastolic Ca2+ leak (Ca2+ sparks) and SR Ca2+ content; Fluo4-AM) were quantified in isolated LV cardiomyocytes without and with the NCX inhibitor SEA0400 (300nM). Results: NXT rats showed stable compensated renal impairment and significantly hypertrophied LV at 8 weeks with a further increase after 24 weeks. LV systolic function (EF, dP/dt) was preserved. End diastolic pressure (EDP) volume relationship was markedly shifted left- and upwards and lung weight were significantly increased, indicating HFPEF with pulmonary congestion. LV cardiomyocytes from NXT showed no significant differences in amplitudes of Ca2+ transients. However, time for early (50%) relaxation of the Ca2+ transients at 8 weeks were significantly prolonged with a further increase after 24 weeks (RT50 17.2±2.9 and 30.8±2.7 vs. 27.6±1.8 and 41.8±2.6 ms; n≥20; p< 0.05). TAU was significantly prolonged at 8 and 24 weeks indicating reduced NCX forward mode activity, while NCX protein expression was upregulated. At 8 weeks, Ca2+ spark frequency tended to be increased (p=0.07) while SR Ca2+ content was unchanged. SEA0400 accelerated Ca2+ transient decay but did not affect Ca2+ spark frequency. At 24 weeks, Ca2+ spark frequency was increased (4.3±0.7 vs. 11.5±1.8 sparks/s/μm3; n≥20; p